Several diseases of the eye result from an underlying genetic cause. For example, in some diseases, a mutation in a protein expressed in cells of the eye alters, or abolishes, the proteins activity resulting in a disease state. In other diseases, the cause may be due to failure of eye cells to produce a particular protein. Because these diseases are due to inactivation, or alteration, of a single protein they are particularly amenable to gene transfer-based therapies. Gene therapy for ocular disease has a set of attractive attributes, including a small tissue target and a closed compartment, which thereby requires a low dose. Additionally the eye is a relatively immune-privileged environment.
One example of an eye disease having a genetic cause is X-linked juvenile retinoschisis (XLRS). XLRS is a neurodevelopmental retinal abnormality that manifests early in life and causes impaired acuity and a propensity to retinal detachment. XLRS is characterized by structural abnormalities in normal lamination of the retinal neuronal and plexiform layers. Clinical examination shows microcysts within the macula, and schisis or internal dissection of the layers of the peripheral retina, (Eksandh L C, Ponjavic V, Ayyagari R, Bingham E L, Hiriyanna K T, Andreasson S, Ehinger B, Sieving P A. 2000. Phenotypic expression of juvenile X-linked retinoschisis in Swedish families with different mutations in the XLRS1 gene. Arch Ophthalmol 118: 1098-1104; Prenner J L, Capone A, Jr., Ciaccia S, Takada Y, Sieving P A, Trese M T. 2006. Congenital X-linked retinoschisis classification system. Retina 26: S61-64) and this is evident by using ocular coherence tomography (Gerth C, Zawadzki R J, Werner J S, Heon E. Retinal morphological changes of patients with X-linked retinoschisis evaluated by Fourier-domain optical coherence tomography. Arch Ophthalmol. 2008; 126:807-11). Impaired retinal synaptic transmission of neural signals causes loss of dark-adapted absolute visual perception. This is evident on clinical electroretinogram (ERG) testing as a characteristic reduction of the b-wave response (from second-order retinal bipolar cells) relative to the photoreceptor a-wave, which frequently gives rise to an ‘electronegative ERG waveform.’ The fragile XLRS retina is more prone to disease related complications, such as vitreous hemorrhage and retinal detachment, and the condition worsens with age. The rate of retinal detachment in the XLRS population is considerably higher than in the general population (10 vs 0.01%, respectively), and the postoperative outcome is much worse.
X-linked juvenile retinoschisis is caused by mutations in the gene-encoding retinoschisin, a 224-amino acid secreted protein that is expressed only by the retina and pineal. Human retinoschisin is composed of a 23-amino acid signal sequence, a 39-amino acid Rs1 domain, a 157-amino acid discoidin domain and a 5-amino acid C-terminal segment. Discoidin domain containing proteins are widely distributed in eukaryotes and mediate a variety of functions, including cell adhesion, cell-extracellular matrix interactions, signal transduction, phagocytosis of apoptotic cells, axon guidance, angiogenesis and blood clotting. Many of these proteins are involved in extracellular matrix or cell binding, although some bind ligands such as vascular endothelial growth factor and semaphorin. Retinoschisin is secreted from retinal neurons as a disulfide-linked homo-octameric complex, which adheres to the cell surface, but its function is not well understood. Biochemical activities attributed to retinoschisin are the binding of b-2-laminin, ab-crystallin, phospholipid, galactose and Na/K ATPase-SARM1 complex. Retinoschisin is first observed in the mouse retina on postnatal day 1. During development, all retinal neurons express retinoschisin after differentiation, beginning with the ganglion cells, which are the first to mature, followed by neurons of each of the more distal layers. From P14 onward, it is strongly expressed in the outer half of the inner nuclear layer and by photoreceptor inner segment. All classes of retinal neurons, except horizontal cell, are shown to be labeled with retinoschisin antibody in adults.
Multiple groups have attempted to use gene-therapy approaches for the treatment of diseases of the eye. For example, several groups have used adeno-associated virus (AAV) vectors expressing retinoschisin to complement the mutations of mice harboring retinoschisin gene deletions. Retinal transduction with these vectors resulted in significant levels of retinoschisin protein in all layers of the retina, and improvement of the disease phenotype, including restoration of the normal positive ERG b-wave and a reduction of the cyst-like structures that are characteristic of the disease. The therapeutic effect was durable and persisted throughout the life of the animal.
In addition to the treatment of X-linked retinoschinosis, other groups have evaluated the clinical use of AAV vectors for the treatment of another X-linked retinopathy, Leber congenital amaurosis (LCA), because of congenital retinal pigment epithelium (RPE) 65 deficiency. AAV vectors expressing RPE65 were administered by subretinal injection to a total of nine subjects with LCA. The nine subjects comprised the collective low-dose cohorts of the three studies, each of which have a dose-escalation design. The majority of the treated subjects showed evidence of improvement in retinal function, visual acuity or reduction in nystagmus despite their relatively advanced state of retinal degeneration.
While the LCA trials used subretinal injection to deliver the vector, this delivery strategy may be problematic for an XLRS trial, as subretinal injection gives geographically localized delivery. Retinoschisin is expressed throughout the retina and optimal treatment of the disease will require transduction of the entire retina. Vector delivery by subretinal injection is limited maximally to about 25% of the retinal area. Although this amount of transduction is sufficient to cover the vicinity of the macula, much of the retina would probably not be transduced, and the untreated area would remain susceptible to retinal detachment and vitreous hemorrhage, which are the major causes of vision loss with this disease. Some additional spread of retinoschisin has been reported in retinas of mice transduced by subretinal injection, but it is not clear how this might scale to human subjects. Subretinal injection of retinas with schisis pathology may be challenging and pose a significant risk to the visual function of the subject. Vitrectomy is usually carried out before subretinal injection. Adhesion of the vitreous to the retina may cause further laminar splitting of the fragile XLRS retina when the surgeon attempts to separate the vitreous from the retina. In addition, the injection itself may also be difficult. If the tip of the injection needle is not positioned deep enough, vector solution may be inadvertently routed into the schisis cavities and exacerbate the intraretinal splitting. An alternative vector administration method would be attractive for XLRS subjects.
In previous work, the inventors described a method for obtaining efficient AAV vector-mediated gene transfer to XLRS retinas without the use of subretinal injection. In that study, all layers of the retinoschisin knockout (Rs1-KO) mouse retina were efficiently transduced with AAV type 2 (AAV2) vectors when administered by simple vitreous injection (Zeng Y, Takada Y, Kjellstrom S, Hiriyanna K, Tanikawa A, Wawrousek E, et al. RS-1 Gene Delivery to an Adult Rs1h Knockout Mouse Model Restores ERG b-Wave with Reversal of the Electronegative Waveform of X-Linked Retinoschisis. Invest Ophthalmol Vis Sci. 2004; 45:3279-85; Kjellstrom S, Bush R A, Zeng Y, Takada Y, Sieving P A. Retinoschisin gene therapy and natural history in the Rs1h-KO mouse: long-term rescue from retinal degeneration. Invest Ophthalmol Vis Sci. 2007; 48:3837-45). However, administration of AAV2 vector leads to a therapy-limiting immune response in the eye, since humans have a high preexisting immunity to AAV2. The inventors developed an AAV vector to complement vitreal administration in humans. The vector was composed of a 3.5-kb human retinoschisin promoter, a human retinoschisin cDNA containing a truncated retinoschisin first intron, the human b-globin polyadenylation site and AAV type 2 (AAV2) inverted terminal repeats, packaged in an AAV type 8 capsid. Intravitreal administration of this vector to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution that was similar to that observed in wild-type retina. Immunolabeling was specific to the retinoschisin-expressing cells of the retina with little or no off-target expression in other eye structures, such as the optic nerve, uveal tissue and cornea.
Thus, the present invention addresses the need for an improved method of delivering therapeutic molecules, such as genes encoding therapeutic proteins, to the eye of an individual in need of such treatment, without eliciting a significant immune response, and provides other benefits as well.